Cell surface receptors are an important class of proteins involved in cellular functioning because they are the primary mediators of cell to cell communication. G protein coupled receptors (GPCRs) are an important category of cell surface receptors. The medical importance of these receptors is evidenced by the fact that more than 60% of all commercially available prescription drugs work by interacting with known GPCRs.
In their resting state, the G proteins, which consist of alpha (α), beta (β) and gamma (γ) subunits, are complexed with the nucleotide guanosine diphosphate (GDP) and are in contact with the receptors to which they are coupled. When a hormone or other first messenger binds to receptor, the receptor changes conformation and this alters its interaction with the G protein. This spurs the α subunit to release GDP, and the more abundant nucleotide guanosine triphosphate (GTP) replaces it, activating the G protein. The G protein then dissociates to separate the α subunit from the still complexed beta and gamma subunits. Either the Gα subunit, or the Gβγ complex, depending on the pathway, interacts with an effector. The effector (which is often an enzyme) in turn converts an inactive precursor molecule into an active “second messenger,” which may diffuse through the cytoplasm, triggering a metabolic cascade. After a few seconds, the Gα converts the GTP to GDP, thereby inactivating itself. The inactivated Gα may then reassociate with the Gβγ complex.
Hundreds, if not thousands, of receptors convey messages through heterotrimeric G proteins, of which at least 17 distinct forms have been isolated. Most G protein-coupled receptors are comprised of a single protein chain that is threaded through the plasma membrane seven times. Such receptors are often referred to as seven-transmembrane receptors (STRs). More than a hundred different GPCRs have been found, including many distinct receptors that bind the same ligand, and there are likely many more GPCRs awaiting discovery. The development of new drug discovery assays to identify novel modulators of GPCRs would be of tremendous benefit.
In recent years drug discovery has been advanced by expression of heterologous receptors in living cells. However, due to the complexity of GPCRs the search for modulators of these receptors have presented particular challenges. For example, there is variability in the sequences of G protein subunits and this variability can influence the efficiency of receptor coupling to subunits. The highest variability has been seen in the α subunit, but several different β and γ structures have also been reported. There are, additionally, several different G protein-dependent effectors.
The mating factor receptors of yeast cells (STE2 and STE3) span the membrane of the yeast cell seven times and are coupled to yeast G proteins. The GPA1, STE4, and STE18 products are the yeast homologues of the α, β and γ subunits of mammalian G proteins, respectively. (Nakafuku et al. 1987. Proc. Natl. Acad. Sci USA 84:2140; Whiteway et al. 1989. Cell. 56:467). Since yeast cells have GPCRs analogous to those found in mammalian cells, experiments have also been undertaken to express functional GPCRs in yeast cells. The use of yeast cells for such expression provides advantages in terms of the ease of manipulating the cells, but also presents particular challenges in achieving efficient coupling and functional integration.
Methods for improving the functional integration of heterologous GPCRs in yeast cells are still needed. The prior art teaches numerous examples of the expression of heterologous receptors in yeast cells which fail to couple to yeast G proteins and thus are not functionally integrated into a yeast signaling pathway. For example Huang et al. teach that the rat M5 receptor, when expressed in yeast cells did not couple to the pheromone response pathway (Huang et al. Biochem. and Biophys. Res. Comm. 1992. 182:1180).
Previous work has demonstrated coupling of heterologous receptors in yeast by the expression of entire foreign G protein subunits in yeast cells (U.S. Pat. No. 5,482,835 to King et al.). Another approach was taken by Kang (1990 Mol. Cell. Biol. 10:2582-2590) who made GPA1-Gα chimeric subunits which comprised large portions, e.g., over 300 amino acids of GPA1. However, the chimeras made by Kang et al. were assayed for their ability to complement a gpal null phenotype (i.e., constitutive activation of the pheromone response pathway) in S. cerevisiae, a situation in which it was desirable to retain a substantial portion of the GPA sequence. Clearly, a method for optimizing the functional integration of a heterologous GPCR into a signaling pathway in a yeast cell expressing such a receptor would be of great value in developing assays to identify receptor agonists and antagonists.